Algal extract comprising sulphated and non-sulphated polyanionic polysaccharides and uses thereof

ABSTRACT

The present invention relates to an algal extract comprising sulphated and non-sulphated polyanionic polysaccharides, to a method for preparing an algal extract as well as to its therapeutic and prophylactic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. Ser. No. 16/017,862, filed onJun. 25, 2018, which was a divisional application of U.S. Ser. No.14/651,345, filed on Jun. 11, 2015, which was a national stageapplication of PCT/EP2013/074105, filed on Nov. 18, 2013, which claimsthe benefit of FR 12 61909, filed on Dec. 11, 2012, each of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an algal extract comprising sulphatedand non-sulphated polyanionic polysaccharides, and to a method forpreparing an algal extract, as well as to its therapeutic andprophylactic applications.

BACKGROUND OF THE INVENTION

The Ulva species (Ulvales, Chlorophyta) are abundant algae found in theintertidal or foreshore area. They colonize hard substrates, anchored byan attachment disc, but certain species may also give rise to freedrifting living forms. Ulva algae are fast growing algae andopportunistic as to space and absorption of nutrients. Their growth inthe water column is particularly observed in eutrophised coastal watersand in lagoons where Ulva sp. proliferates in the form of “green tides”(Fletcher, 1996). Often mass production and mass strandings, producingnoxious gases when they accumulate, result from this (Morand and Briand,1996). Up till now, this biomass had very little added value and themeans for using it except for compost (Mazé et al. 1993, Cuomo et al.1995), for producing methane (Briand and Morand, 1997), for human foodconsumption (Pérez, 1997) or as a basis for paper (Nicolucci andMonegato 1993) might give the possibility of making the most out oftheir specific properties.

At the present time, the use of green algae is essentially based on usesgiving the possibility of opening new routes for removing these algaeconsidered as undesirable. The contemplated application routes concernthe production of biogas (Briand & Morand 1997, Morand & Briand 1999,Morand et al., 2006), biomaterials (bioplastics) (Chiellini et al.,2008), biodegradable cups (Sassi et al., 2008), papers and cardboardsfor packaging (Nicolucci & Monegata et al., 1993), chargenano-composites (Demais et al., 2006a, 2006b), animal nutrition:detoxifying feed for animals (Demais et al., 2006), proteins andappetence factors for fish farming (DeBose et al., 2008, Kut Guroy etal., 2007) and cosmetics (surfactants) (Ranson et al., 2008).

The chemical and physico-chemical properties of the polysaccharidescontained in these algae make them attractive candidates as novelfunctional and biologically active polymers in the fields of human andanimal consumption, of pharmaceutics, of chemistry, of fish farming andagriculture (Robic A, et al. Carbohydrate Polymers 77 (2009) 206-216;Lahaye M Robic A, Biomacromolecules Vol 8 No. 6, 2007).

DETAILED DESCRIPTION OF THE INVENTION

According to one of these aspects, the present invention relates to anextract from algae of the order of the Ulvales, in particular an extractfrom green algae of the Ulva type, comprising sulphated andnon-sulphated polyanionic polysaccharides, the size of which is lessthan or equal to 50 kDa.

In particular, said polysaccharides of the algal extract according topresent invention comprise mannose and/or arabinose. More particularly,said polysaccharides comprise at least 0.005% of mannose and/or at least0.005% of arabinose, by weight based on the weight of the total drymaterial of the algal extract, notably at least 0.01% of mannose and/orat least 0.01% of arabinose. Still more particularly, saidpolysaccharides comprise mannose in an amount ranging from 0.01 to 0.20%and arabinose in an amount ranging from 0.01 to 0.5%, by weight based onthe weight of the total dry material of the algal extract, notablymannose in an amount ranging from 0.03 to 0.15% and arabinose in anamount ranging from 0.01 to 0.2%.

In particular, said polysaccharides further comprise:

-   -   galactose;    -   glucose;    -   rhamnose;    -   xylose; and    -   glucuronic acid.

Still more particularly, said polysaccharides comprise:

-   -   from 0.05 to 0.5% of galactose, notably from 0.1 to 0.4%;    -   from 0.005 to 0.05% of glucose, notably from 0.01 to 0.03%;    -   from 2 to 15% of rhamnose, notably from 5 to 10%;    -   from 0.1 to 1% of xylose, notably from 0.3 to 0.7%; and    -   from 1 to 7% of glucuronic acid, notably from 1 to 5%;        by weight based on the weight of the total dry material of the        algal extract.

Thus, mention may for example be made of an algal extract according tothe invention comprising:

mannose;

arabinose;

galactose;

glucose;

rhamnose;

xylose; and

glucuronic acid.

More particularly mention may for example be made of an algal extractaccording to the invention comprising:

from 0.01 to 0.20% of mannose, notably from 0.03 to 0.15%;

from 0.01 to 0.5% of arabinose, notably from 0.01 to 0.2%;

from 0.05 to 0.5% of galactose, notably from 0.1 to 0.4%;

from 0.005 to 0.05% of glucose, notably from 0.01 to 0.03%;

from 2 to 15% of rhamnose, notably from 5 to 10%;

from 0.1 to 1% of xylose, notably from 0.3 to 0.7%; and

from 1 to 7% of glucuronic acid, notably from 1 to 5%;

by weight based on the weight of the total dry material of the algalextract.Still more particularly mention may for example be made of an algalextract according to the invention comprising:

0.09% of mannose;

0.1% of arabinose;

0.3% of galactose;

0.02% of glucose;

8.1% of rhamnose;

0.5% of xylose; and

2.6% of glucuronic acid;

by weight based on the weight of the total dry material of the algalextract.

More particularly, the algal extract according to the present inventioncomprises sulphated and non-sulphated polyanionic polysaccharides forwhich the size is less than 40, 30, 20 or 15 kDa. Still moreparticularly, the sulphated and non-sulphated polyanionicpolysaccharides of the algal extract according to the invention has asize of less than or equal to 15 kDa.

A dalton (Da) is a mass unit defined as being equal to one-twelfth ofthe mass of a carbon 12 atom, a mass which will subsequently beestimated from a mixture of several isotopes (mainly carbon 12 andcarbon 13, respectively having 6 and 7 neutrons in addition to the 6protons like any carbon atom). One dalton is, with quite good accuracy,the mass of a hydrogen atom, the exact value being 1.00794 amu (atomicmass unit). The kilodalton (kDa) is equal to 1,000 Da.

Within the scope of the present invention, the masses mentioned in kDaare determined by any method usually used by one skilled in the art, inparticular the masses of the sulphated and non-sulphated polyanionicpolysaccharides of the algal extracts according to the present inventionmay be discriminated by ultrafiltration on membranes only lettingthrough molecules of predetermined sizes.

By “green algae of the Ulva type” are meant the green algae grouped inthe genus Ulva from the family of Ulvaceae, of the order of the ulvales.Mention may notably be made of the following species and sub-species:Ulva acanthophora, Ulva anandii, Ulva angusta, Ulva arasakii, Ulvaarmoricana, Ulva atroviridis, Ulva attenuata, Ulva beytensis, Ulvabifrons, Ulva brevistipitata, Ulva bulbosa, Ulva burmanica, Ulvabyssoides, Ulva californica, Ulva chaetomorphoides, Ulva clathrata, Ulvacoccinea, Ulva compressa, Ulva conglobata, Ulva cornucopiae, Ulvacornuta, Ulva covelongensis, Ulva crassa, Ulva crassimembrana, Ulvacurvata, Ulva dactylifera, Ulva denticulata, Ulva elegans, Ulvaelminthoides, Ulva enteromorpha, Ulva erecta, Ulva expansa, Ulvafasciata, Ulva fenestrata, Ulva flexuosa, Ulva gelatinosa, Ulvageminoidea, Ulva gigantea, Ulva grandis, Ulva hendayensis, Ulvahookeriana, Ulva hopkirkii, Ulva indica, Ulva intestinalis, Ulvaintestinaloides, Ulva intricata, Ulva intybacea, Ulva javanica, Ulvakylinii, Ulva lactuca, Ulva lactucaefolia, Ulva laetevirens, Ulvalaingfi, Ulva linearis, Ulva lingulata, Ulva linkiana, Ulva linza, Ulvalippii, Ulva litoralis, Ulva littorea, Ulva lobata, Ulva lubrica, Ulvamarginata, Ulva micrococca, Ulva myriotrema, Ulva neapolitana, Ulvanematoidea, Ulva ohnoi, Ulva olivacea, Ulva olivaceum, Ulva pacifica,Ulva papenfussii, Ulva paradoxa, Ulva parva, Ulva parvula, Ulvapatengensis, Ulva percursa, Ulva pertusa, Ulva phyllosa, Ulvapopenguinensis, Ulva porrifolia, Ulva procera, Ulva profunda, Ulvaprolifera, Ulva pseudocurvata, Ulva pseudolinza, Ulva pulchra, Ulvapurpurascens, Ulva quilonensis, Ulva radiata, Ulva ralfsii, Ulvaranunculata, Ulva reticulata, Ulva rhacodes, Ulva rigida, Ulvarotundata, Ulva rubens, Ulva saifullahii, Ulva scagelii, Ulvascandinavica, Ulva sericea, Ulva serrata, Ulva simplex, Ulva sorensenii,Ulva spinulosa, Ulva stenophylla, Ulva stipitata, Ulva sublittoralis,Ulva subulata, Ulva taeniata, Ulva tenera, Ulva tetragona, Ulva torta,Ulva tuberosa, Ulva umbilicata, Ulva uncialis, Ulva uncinata, Ulvausneoides, Ulva utricularis, Ulva utriculosa, Ulva uvoides, Ulvaventricosa.

More particularly, the algal extract according to the invention is anextract from algae of the Ulva armoricana species.

According to an embodiment of the invention, the algal extractcomprises:

-   -   from 10 to 50% of carbon;    -   from 1 to 10% of hydrogen;    -   from 1 to 5% of nitrogen;    -   from 20 to 50% of oxygen; and    -   from 1 to 15% of sulphur;

in percent by mass of the total dry material of the algal extract.

Still more particularly, the algal extract comprises:

-   -   from 15 to 30% of carbon;    -   from 3 to 6% of hydrogen;    -   from 1 to 3% of nitrogen;    -   from 25 to 40% of oxygen; and    -   from 2.5 to 10% of sulphur;

in mass percentage of the total dry material of the algal extract.

The other chemical elements present in the dry material of the extractare notably represented by the minerals (Ca, K, Na, Mg, Al, Cl, I, P,Fe, etc).

More particularly, the algal extract according to the invention ischaracterized by the ¹H NMR spectrum shown in FIG. 1.

This ¹H NMR spectrum was recorded at 298 K on a Bruker Avance 500spectrometer equipped with an inverse TCI cryogenic probe 5 mm¹H/¹³C/¹⁵N. Before the analysis, the samples were dissolved in 99.97% ofD₂O atoms. The chemical shifts are expressed in ppm relatively to anexternal standard (trimethylsilylpropionic acid). No suppression of theHOD signal was achieved.

According to another of these aspects, the present invention relates toa method for producing an extract from algae of the order of ulvales, inparticular an extract from green algae of the Ulva type, wherein:

-   -   a) the algae are washed and freed of sand;    -   b) said algae are crushed;    -   c) the solid phase of the crushed product is separated from its        liquid phase;    -   d) said liquid phase is clarified;    -   e) the juice obtained in step d) is ultrafiltrated; and    -   f) the filtration juice obtained in step e) is concentrated and        then dried.

According to an embodiment of the method according to the presentinvention, the algae are washed with fresh water.

Sand may be removed from them by any means made available to one skilledin the art.

Said algae are then milled, notably by means of a milling machine, suchas for example a refiner or a cutter.

Subsequently, the solid phase of the milled product, the marc, isseparated from its liquid phase, the juice, by pressing the milledproduct, for example by means of a belt or plate press or bycentrifugation.

By “juice”, is meant the cytoplasm juice which includes the wallstructure of the double structure of the cells of the algae.

The obtained liquid phase is then clarified, for example with aclarifier with disk stacks, or by centrifugation, decantation orfiltration (for example with a pocket or a plate).

The obtained juice is then ultrafiltrated.

According to an embodiment of the method according to the presentinvention, ultrafiltration is carried out on a membrane of less than 50kDa, notably on a 40, 30, 20 or 15 kDa membrane. More particularly, themembrane will be a 15 kDa membrane.

This membrane may for example be a ceramic membrane or an organicmembrane. More particularly, the membrane will be a ceramic membrane.

The obtained filtration juice may then be concentrated, for example byreverse osmosis, evaporation or precipitation, and then for exampledried by freeze-drying or atomization.

Optionally, the obtained extract may then again be crushed, in order toobtain a homogeneous powder in terms of grain size.

According to one of these aspects, the method according to the inventionpartly takes place at room temperature. By room temperature is meant atemperature comprised between 5 and 25° C.

According to another of these aspects, the method according to theinvention partly takes place at a temperature comprised between 4 and10° C., this in order to avoid microbial growth.

This method differs from most methods described in the prior art becauseof the absence of a step involving precipitation of the algal extract.It is also distinguished from the previous extraction methods by theabsence of any use of solvents, in particular organic solvents, whichrepresents a major advantage from an ecological point of view.

According to another of these aspects, the present invention relates toan algal extract which may be obtained by the method as describedearlier.

According to another of these aspects, the present invention relates toan algal extract as defined above which may be obtained by the method asdescribed earlier.

According to one of these aspects, the present invention also relates toa pharmaceutical composition comprising an algal extract as describedearlier as well as to a pharmaceutically acceptable excipient.

According to one of these aspects, the present invention also relates toan algal extract as described earlier for its use as a drug.

According to one of these aspects, the present invention also relates toan algal extract as described earlier for its use in preventing and/ortreating neurological disorders, depression, stress, anxiety, as anagent allowing improvement of memory notably in preventing and/ortreating ageing or Alzheimer's disease, in preventing and/or treatingneuropathic and inflammatory chronic pain, skin pain (of the skin, ofsubcutaneous tissues and associated organs) or body pain.

According to another of these aspects, the present invention alsorelates to the use of an algal extract as described earlier as a drug.

According to one of these aspects, the present invention also relates tothe use of an algal extract as described earlier in preventing and/ortreating neurological disorders, depression, stress, anxiety, as anagent allowing improvement of memory notably in preventing and/ortreating ageing or Alzheimer's disease, in preventing and/or treatingneuropathic and inflammatory chronic pain, cutaneous pain (of the skin,subcutaneous tissues and associated organs) or body pain.

By “neurological disorders” are meant diseases of the nervous system, inparticular of the brain. Among these diseases, mention may notably bemade of brain pathologies such as Parkinson's disease, multiplesclerosis, Alzheimer's disease, dementia, migraines, headaches.

By “depression” is meant a mood disorder essentially characterized by acondition with loss of motivation or vital momentum in an individual,either associated or not with different symptoms. The mostcharacteristic symptoms are a loss of hope, of desire, of self-esteem.Other signs may occur, such as fatigue, sadness, negative thoughts, darkthoughts, suicidal intentions, anxiety or fear or in certain extremelyrare cases, hallucinations.

This term of depression is again found under the names of “recurrentdepressive disorder”, “nervous breakdown”, “clinical depression”,“unipolar depression”, “major depressive and characterized episode” orfurther “depressive syndrome”. The term of “depressions” designates thewhole of the types of depression. The terms of “depressivity” or“depressive feeling” are also used. Common language also mentions“feeling low”, which has similar symptoms, but more attenuated.

By “stress”, is meant the whole of the responses of an organism subjectto pressures or constraints from its environment. These responses alwaysdepend on the perception which the individual has of the pressures whichhe/she feels. This is a complex sequence of events causing physiologicaland psychosomatic responses.

By “anxiety”, is meant a psychological and physiological conditioncharacterized by somatic, emotional, cognitive and behaviouralcomponents. In the absence or in the presence of psychological stress,anxiety may generate feelings of fear, restlessness, difficulty andworries. Anxiety is considered as a normal reaction in a stressingsituation. When anxiety becomes excessive, it may be classified underthe name of “anxiety disorder”.

Thus, an algal extract according to the present invention may be usedboth in veterinary applications, such as for example for preventingand/or treating stress in livestock and pets or as an anti-depressantagent for livestock or pets, and in applications intended for humans,for example via food supplements with a health purpose without anysecondary effects or a drug, in order for example to prevent and/ortreat disorders related to stress, to anxiety, to depression, as well asan agent allowing improvement in memory, notably in students, forcombating ageing or further within the scope of treating Alzheimer'sdisease.

The pharmaceutically acceptable excipients are selected according to thepharmaceutical form and to the desired administration route, from amongusual excipients which are known to one skilled in the art.

In the pharmaceutical compositions of the present invention for oral,sublingual, subcutaneous, intramuscular, intravenous, topical, local,intratracheal, intranasal, transdermal or rectal administration, thealgal extract as defined above may be administered as unit doses, mixedwith conventional pharmaceutical excipients to animals and to humanbeings, for preventing or treating the aforementioned disorders.

The suitable administration forms comprise oral forms such as tablets,soft or hard gelatin capsules, powders, granules or oral solutions orsuspensions, sublingual, buccal, intratracheal, intraocular, intranasaladministration forms, by inhalation, topical, parenteral such astransdermal, subcutaneous, intramuscular or intravenous administrationforms, rectal administration forms and implants. For topicalapplication, the compounds according to the invention may be used increams, gels, ointments or lotions.

When a solid composition such as tablets is prepared, the main activeingredient may be mixed with a pharmaceutical carrier such as gelatin,starch, lactose, magnesium stearate, talcum, gum arabic or the like.

The tablets may also be coated with saccharose, a cellulose derivative,or other suitable materials or further they may be treated so that theyhave a prolonged or delayed activity and continuously release apredetermined amount of active ingredient.

A preparation as gelatin capsules may for example be obtained by mixingthe active ingredients with a diluent and by pouring the obtainedmixture into soft or hard gelatin capsules.

The pharmaceutical compositions containing an algal extract according tothe invention, may also appear in liquid from, for example, assolutions, emulsions, suspensions or syrups, and notably in a suitableform for oral or intranasal administration for example. The suitableliquid supports may for example be water, organic solvents such asglycerol or glycols, as well as their mixtures, in varied proportions,in water.

A preparation as a syrup or elixir or for administration in drop formmay also contain the active ingredient together with an acaloricsweetener for example, methylparabene or propylparabene as anantiseptic, as well as an agent providing taste and a suitable colouringagent.

The powders or the granules dispersible in water may for example containthe active ingredient mixed with dispersion agents or wetting agents, orsuspension agents, such as polyvinylpyrrolidone, also with sweeteners orflavour correctors.

The dose administered daily in humans may attain 0.5 to 25 mg/kg, in oneor several takings, in particular from 1.5 to 18 mg/kg, and still moreparticularly from 3 to 15 mg/kg. The daily administered dose in animalsmay attain 5 to 150 mg/kg, in one or several takings, in particular from10 to 100 mg/kg and even more particularly from 20 to 80 mg/kg.Generally, the daily dose of the algal extract according to theinvention will be the smallest effective dose of the algal extractcapable of producing a therapeutic effect.

By “effective dose”, is meant any amount of a composition which improvesone or several of the characteristic parameters of the disease or of thedisorder to be treated.

There may be particular cases where higher or lower dosages aresuitable; such dosages do not depart from the scope of the presentinvention. According to customary practice, the suitable dosage for eachpatient, human or animal is determined by the physician or theveterinary surgeon according to the administration route, the weight andthe response of said patient.

According to another of its aspects, an algal extract according to thepresent invention may be used for preventing and/or treating theaforementioned pathologies in a food composition.

By “food composition” is for example meant any type of nutraceutical, offood products as a yogurt or a drink, notably a dairy drink, any type ofraw material, technological additive or auxiliary, in the form ofpre-mixes, either medicinal or not, intended to be incorporated intofoodstuffs, any type of full or supplement foodstuffs, intended forhumans or animals.

The present invention, according to another of its aspects, also relatesto a method for treating the pathologies indicated above which comprisesadministration to a patient of an effective dose of an algal extractaccording to the invention.

The present invention will be illustrated in more detail by the figuresand examples below which do not limit the scope thereof.

FIGURES

FIG. 1: ¹H NMR spectrum of an algal extract (AE) according to thepresent invention.

FIG. 2: Chromatogram obtained with the algal extract according to theinvention separated on two Shodex 802 and 803 columns.

FIG. 3: Chromatogram obtained after analysis of trimethylsilylatedderivatives of the sample of the algal extract according to theinvention by gas chromatography. With Ara: Arabinose; Gal: Galactose;Glc: Glucose; Xyl: Xylose; Man: Mannose; Rha: Rhamnose, GlcA: Glucuronicacid.

FIG. 4: Effects of AE on the number of entries into the open arms in theelevated plus-maze test.

FIG. 5: Effects of AE on the time spent in the open arms in the elevatedplus-maze test.

FIG. 6: Effects of AE on the total number of cumulated pressing actionson both levers in the learning test for avoiding an aversive lightstimulus (ALSAR for Aversive Light Stimulus Avoidance Response).

FIG. 7: Effects of AE on the total number of pressing actions on theactive levers (LA) and inactive levers (LI) at 5 minutes in the ALSARtest.

FIG. 8: Effects of AE on the total number of pressing actions on theactive levers (LA) and inactive levers (LI) at 10 minutes in the ALSARtest.

FIG. 9: Effects of AE on the total number of pressing actions on theactive levers (LA) and inactive levers (LI) at 15 minutes in the ALSARtest.

FIG. 10: Effects of AE on the total number of pressing actions on theactive levers (LA) and inactive levers (LI) at 20 minutes in the ALSARtest.

FIG. 11: Effects of AE on the duration of immobility(ies) in thepre-test of behavioural despair at D14 (s, Average±SME).

FIG. 12: Effects of AE on the duration of immobility(ies) in thebehavioural despair test at D15 (s, Average±SME).

EXAMPLES Example 1: Preparation of an Algal Extract According to theInvention

One metric ton of fresh, raw, green algae of the Ulva type, is washedwith fresh water and sand is removed by means of a machine for washingalgae.

Unless indicated otherwise, the steps of the method are carried out atroom temperature.

The algae (1 metric ton of drained algae with 8% dry material) are thencrushed into fine particles by means of an industrial refiner (Inotecbrand type “I175CDI-75D”). By “fine particles” are meant particles forwhich the size is comprised between 50 and 1,000 nm, with twopopulations, the first for which the sizes are comprised between 50 and200 nm, the second for which the sizes are comprised between 600 and1,000 nm.

The crushed material was then pressed by means of an industrial beltpress of the brand Flottweg type “B FRU 800 HK”, at a throughput ofabout 1 metric ton/hour.

This step allows separation of the solid phase (marc) from the liquidphase (juice). The yield of obtained juice is 75%.

The 750 kg of raw juice obtained are then clarified by means of aclarifier with disc stacks of the brand Flottweg type “AC 2000”.

710 kg of a clear juice with 3.10% of dry mass (95 to 98% mass yield)and a cream (2 to 5% by mass) are thereby obtained.

Subsequently, the clear juice is ultrafiltrated on a ceramic membrane(Tami Industries) of 15 kDa.

A permeate and a retentate are thereby obtained. The permeate is keptuntil 640 kg of filtration juice (91% of volume yield) with 2.2% of drymaterial are obtained.

The filtration juice (permeate) is then dried by freeze-drying afterconcentration by evaporation.

The concentration is achieved on a simple effect evaporator (EVA 1000,Pignat) with the following parameters: forced reflow, supply flow rate10 L/h, vapour pressure of 1 bar, vacuum pressure of 0.3 bars andevaporation temperature of 90° C.

A first concentration is achieved with an evaporated water flow of 8 L/hand the Brix degree rises from 5.5 (equal to a dry materialconcentration of 4.5%) to 14.7. This solution is then concentrated asecond time with an evaporated water flow of 5-6 L/h and the Brix degreerises up to 34. The dry material concentration of the solution isdetermined at 38.4%.

Freeze-drying is then carried out by means of a Bioblock scientificapparatus (version CHRIST alpha 1-4 LSC) at a freezing temperature of−80° C. which is also the minimum temperature during this step.

The obtained powder is then crushed with a planetary milling machineMiniMill of the Philips brand. The product was introduced into millingbowls (10 g of product in each milling bowl with 4 zirconia balls). Thewhole was set into rotation for 15 minutes at speed 10.

14 kg of an algal extract powder were thereby obtained.

Example 2: Determination of the Size of Sulphated and Non-SulphatedPolyanionic Polysaccharides of an Algal Extract According to theInvention by GPC (Gel Permeation Chromatography)

The algal extract according to the invention and prepared according toExample 1 is ultrafiltrated on a 1,000 Da membrane and is dissolved at aconcentration of 0.5 g/L in water. It is then injected on two Shodex 802and 803 columns placed in series (fractionation domain of the 802column: 4·10³ Da and of the 803 column: 1.7·10⁵ Da). The eluent used is0.1 M sodium nitrate with 0.2% sodium azide at a flow rate of 0.5ml/min. Detection is achieved via a Wyatt refractometre and an 18-anglelight scattering detector Wyatt. The do/dc are assumed to be equal to0.150 ml/g.

The chromatogram detected by the refractometre is shown in FIG. 2.

An average size of 4.4 kDa of the polysaccharides from an algal extractaccording to the invention is obtained.

Example 3: Determination of the Composition of an Algal ExtractAccording to the Invention

The algal extract according to the invention and prepared according toExample 1 is purified by front ultrafiltration in amicon cells operatingwith stirring. A regenerated cellulose membrane with a cut-off thresholdof 1,000 Da is used. 572.1 mg of a sample of algal extract according tothe invention are dissolved in 150 ml of ultrapure milli-Q water. Fivelitres of water are used for removing all the molecules with a mass ofless than 1,000 Da. The retentate is freeze-dried. 117 mg of sample areweighed. The ultrafiltration yield is therefore 20.5% (w/w). Thefollowing analyses were carried out on ultrafiltrated samples.

The ratio of the monosaccharides making up the polysaccharides of thealgal extract according to the invention is determined according to theKemerling method (Kemerling et al., 1975) modified by Montreuil(Montreuil et al., 1986). The identification and dosage of themonosaccharides requires hydrolysis by methanolysis of the polymer, inorder to only obtain monomers. The glycoside residues are thentrimethylsilylated in order to make them volatile. They are thusidentified and assayed by gas chromatography in the form ofo-trimethylsilylated methylglycosides.

The following reagents are used:

A 3N methanol/HCl solution (Supelco);

Silver carbonate;

Myo-inositol;

Pyridine;

Sylon BFT (BSTFA+TMCS 99:1) reagent (Supelco); and

Dichloromethane.

The operating procedure is the following: 400 μg of the algal extractaccording to the invention prepared as mentioned above and 50 μg ofmyo-inositol are placed in a dry bath in the presence of 500 μl of a 3 Nmethanol/hydrochloric acid mixture (Supelco) for 4 hours at 100° C.After cooling down to room temperature, the methanolysate is neutralizedwith silver carbonate. The samples are centrifuged for 15 minutes at3,000 rpm and the supernatant is evaporated under a nitrogen jet. Thecompounds are then dissolved in 80 μl of pyridine and incubated for 25minutes at 80° C. with 80 μl of sylon (BSTFA: TMCS, 99:1, Supelco).After gentle evaporation of the excess reagents under a nitrogen jet,the trimethylsilylated methylglycosides are taken up in 500 μl ofdichloromethane and then injected into gas chromatography (injectioninto a column, FID detector: flame ionization). The carrier gas isnitrogen. The column, of the HP-5MS type (30 m, inner diameter 0.25 mm),is apolar. The programme for the rise in temperature is the following:120° C. maintained for 1 minute, and then a gradient of 1.5° C./min upto 180° C., followed by a gradient of 2° C./min up to 200° C.

Each monosaccharide is identified by comparing its relative retentiontimes relatively to the internal standard, with those of puremonosaccharides treated under the same conditions. A responsecoefficient is calculated for each monosaccharide relatively to theinternal standard in order to define the proportion of eachmonosaccharide within the polysaccharides of the algal extract accordingto the invention.

The obtained results are shown in Table 1 below and in FIG. 3.

TABLE 1 Composition of the algal extract according to the inventionobtained after analysis of trimethylsilylated derivatives by gaschromatography, expressed by weight based on the total weight of thealgal extract; with Ara: Arabinose; Gal: Galactose; Glc: Glucose; Xyl:Xylose; Man: Mannose; Rha: Rhamnose, GlcA: Glucuronic Acid. % by weightof the % by weight of the Sample ultrafiltrate Ultrafiltration yield rawextract Ara 0.6 20.50% 0.123 Gal 1.3 20.50% 0.267 Glc 0.1 20.50% 0.021Xyl 2.45 20.50% 0.502 Man 0.45 20.50% 0.092 Rha 39.6 20.50% 8.118 GlcA12.9 20.50% 2.645

Example 4: Evaluation of the Effects of the Algal Extract (AE) Through aFunctional Battery of Tests and of Additional Tests (Open Field,Elevated-Plus Maze, Test for Avoiding Aversive Stimulus)

The effects of an algal extract according to the invention (AE)administered orally at doses of 20, 40 and 80 mg/kg for 14 days wereevaluated in adult male Wistar rats.

The effects of AE were evaluated on groups of 6 rats as compared with acontrol batch treated with the carrier (spring water) through afunctional battery of tests (FOB). Additional tests, open field,elevated-plus maze and the test for avoiding an aversive light stimulus,were conducted for completing the evaluation of the effects of AE.

The rats used were treated according to the ethical rules dictated byASAB and the Canadian Council for Animal Protection.

Equipment and Methods Animals

Twenty four (24) male Wistar Crl:WI (Han) rats (Charles RiverLaboratories, 69-St-Germain sur l'Arbresle, France) of 60 days of agewere used. Upon receiving them, the rats were marked and distributed ingroups of 2 in polycarbonate cages of the F type (48×27×20 cm, U.A.R.,91-Epinay-Sur-Orge, France). The animals were confined in anair-conditioned animal housing facility, at a temperature of 22±2° C.and with 50±20% hygrometry. The rats had standard 2016 feed (Mucedolafor Harlan, Milan, Italy) and drink ad libitum and were subject to areverse light-darkness cycle of 12 hours (light from 8 p.m. to 8 a.m.).

After getting used to the laboratory conditions for one week, the ratsare weighed and randomly distributed into 4 treatment groups (n=6).

In order to avoid possible interferences between the various products,the rats of a same cage all received the same treatment. The rats of thedifferent groups were all handled in the same way and under the sameconditions.

The FOB Test

The following equipment was used in this test:

a transparent observation cage;

a suspension device;

a gripping device;

an open field for locomotor and exploratory activity;

an auditive stimulation apparatus emitting a standard sound stimulus;

a stylus for various tactile stimulations;

a digital thermometer.

The FOB test was conducted as a blind test before administering theproduct (T0) and after a period of 14 days of treatment (T14) via anoral route at doses of 20, 40 and 80 mg/kg.

The test included three observation phases:

a direct observation phase during which the animal is not disturbed;

an active observation phase during which the animal was handled;

a phase dedicated to evaluating the behavioural reactions of the animalsfollowing reactivity tests.

The recorded variables were the following:

Behavioural effects: spontaneous locomotor activity, locomotor behaviourdisorders, flight reaction to being touched, irritability, causedaggressive and freezing behaviours, drowsiness, micturition anddefecation, sensorimotor responses (visual placement, pinching the paw,pinching the tail and reaction to a sound stimulus).

Neurological effects: pupil response, palpebral reflex, pelvicelevation, position of the tail, muscular tonicity of the limbs and ofthe abdomen, rollover test, gripping test, trembling and piloerection.

Physiological effects: salivation, lacrimation, diarrhoea, rectaltemperature, cardiac-respiratory rhythm.

Mortality.

The following tests were conducted after two weeks of administration ofthe products:

Elevated Plus Maze Test (LCS)

The experimental device, with the shape of a cross, is elevated to 80 cmabove the ground. It comprises four arms with a length of 50 cm and awidth of 15 cm. Two opposite arms are closed by vertical walls with aheight of 40 cm, the two other arms being open to empty space, and,consequently represent anxiogenic locations. On day 18, one hour afteradministration of the treatment, the animal was placed at the centre ofthe cross of the device and was able to freely access the whole of thefour arms. The behaviour of the animal was recorded by means of theANYMAZE system for 5 minutes. The variables studied in this test werethe number of entries into the open arms, as well as the time spent inthese open arms. A small number of entries and a short time spent in theopen arms are considered as indicating anxiety (Lister, 1987).

Aversive Light Stimulus Avoidance Response (ALSAR) Test

This model uses aversion of the rat for a strongly illuminatedenvironment. The rat learns to master its aversive light environmentwithin the scope of conditioning which operates by pressing on an activelever in order to obtain darkness periods of 30 seconds as a positivereinforcement.

The experimental device consists in a strongly illuminated isolated cage(50×40×37 cm), and including two levers: one active one, allowing whenit is actuated, 30 seconds of darkness to be obtained followed by returnof the light, while the other lever is inactive (does not cause anypositive reinforcement). The pressing actions on the active lever,during the darkness period do not provide additional darkness periods.The rat is placed in the cage for 20 minutes and the number of pressingactions on each lever is counted during the experimentation.

The test battery, consisting of 4 conditioning devices is entirelyautomated and controlled via a computer. Thus, no experimenter ispresent in the room during the test.

After 18 days of administration of the products to be tested, the ratswere tested on a cognitive level for acquiring training within the scopeof conditioning operating in the ALSAR model.

The recorded variables were the total number of pressing actions on theactive (LA) and inactive (LI) levers and the numbers of pressing actionson each of both levers during the light phase.

The numbers of active and inactive pressing actions allowed evaluationof the level of manipulatory activity during the test session. Acquiringtraining (discrimination between both levers) was evaluated by comparingthe number of pressure actions on each of the two levers during thelight phase (LA vs. LI).

Product

The product is an AE (algal extract) as a powder representing afreeze-dried and crushed concentrated fraction of a water-soluble algalextract prepared according to Example no. 1. The AE is dissolved inspring water and administered orally 60 minutes before the FOB tests andthe additional behavioural tests by means of an intragastric feedingprobe.

Statistical Analysis

The Kruskal-Wallis test was applied, followed in the cases ofheterogeneity, by comparisons by means of the Mann-Whitney U test forcomparing the treated groups with the control group.

Wilcoxon's test was used for two-by-two comparisons of the repeated FOBmeasurements, between T0 and T14 in each of the groups and fordiscriminating between both ALSAR levers in an illuminated environment.

The statistical and graphical processing operations were carried out bymeans of the software package StatvieW 5 (SAS Institute, Inc., USA).

Results FOB Test

Comparisons of the Groups Treated with the Carrier Group in the FOBs atT0 and T14

In the FOB at T0, before administering the treatment, the results of allthe studied rat groups are homogeneous (Table 2, Table 3).

In the FOB tests at T14 (after 14 days of administration of thetreatments), the Kruskal-Wallis test showed homogeneity of the resultswithin many variables from among the different treatment groups (Table2).

On the other hand, on certain studied variables, the AE at certain dosesshowed significant effects as compared with the carrier (Table 3).

The main variables modified by daily administration of AE for two weeksare:

-   -   The behaviour of the rats and their spontaneous locomotor        activity in a housing cage: the rats treated with 3 doses are        more active and more alert,    -   The behaviour of the rats in the observation cage: there also,        the rats are more active and more alert with 2 stronger doses,    -   Interest for a shown object: at the 3 doses, AE seems to have a        stimulating effect on the curiosity of the rats and their        interest for novelty (increase in the exploratory activity,        hedonism, antidepressant effect),    -   The flight reactions of the rats upon approaching a finger or        upon touching them are significantly reduced by AE at the 3        doses, which comes under an anti-stress/anxiolytic effect of AE,    -   The reactions to pinching of the tail and to pinching of the paw        are also significantly reduced at the 3 doses. These lowered        reactions come under an anti-stress/anxiolytic effect of AE        and/or an antalgic effect,    -   The cardiac and respiratory frequencies of the rats seem to be        lower than in rats treated with AE at the 3 doses, these come        under an anti-stress/anxiolytic effect of AE,    -   Agitation, nervosity and irritability of the rats were        significantly lowered under administration of AE at the 2        strongest doses, coming under a calming, anti-stress and/or        anti-aggressivity effect.

TABLE 2 State of the variables before (T0) and after (T14)administration of AE for 14 days at the doses of 20, 40 and 80 mg/kg,p.o. FOB session T0 T14 Groups AE AE AE AE AE AE Items Carrier 20 40 80Carrier 20 40 80 Displacement latency in H(ddl3) = 5.14; H(ddl3) = 4.10;the arena P = 0.16 P = 0.25 Locomotor activity H(ddl3) = 3.06; H(ddl3) =2.43; (spaces crossed) P = 0.38 P = 0.49 Locomotor activity H(ddl3) =4.91; H(ddl3) = 2.96; (straightening up) P = 0.18 P = 0.40 Awaken ordrowsy H(ddl3) = 3.00; H(ddl3) = 0.00; condition P = 0.39 P = 1.00Spaces crossed on an H(ddl3) = 3.00; H(ddl3) = 3.94; open branch P =0.39 P = 0.27 Rollover reaction H(ddl3) = 0.00; H(ddl3) = 0.00; P = 1.00P = 1.00 Gripping reaction H(ddl3) = 1.11; H(ddl3) = 2.09; P = 0.77 P =0.55 Suspension test H(ddl3) = 3.15; H(ddl3) = 1.89; P = 0.37 P = 0.59Jump reaction to a H(ddl3) = 4.26; H(ddl3) = 2.13; sound stimulus P =0.23 P = 0.55 Caused aggressivity H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P = 1.00 General muscular H(ddl3) = 3.34; H(ddl3) = 6.39; tonicityP = 0.34 P = 0.10 Limb muscular tonicity H(ddl3) = 4.60; H(ddl3) = 3.29;P = 0.20 P = 0.35 Visual placement H(ddl3) = 5.63; H(ddl3) = 1.97;reaction on the rod P = 0.13 P = 0.58 Shrieks H(ddl3) = 2.30; H(ddl3) =4.31; P = 0.51 P = 0.23 Defecation H(ddl3) = 5.43; H(ddl3) = 3.22; P =0.14 P = 0.36 Micturition H(ddl3) = 6.27; H(ddl3) = 2.30; P = 0.10 P =0.51 Convulsions H(ddl3) = 0.00; H(ddl3) = 0.00; P = 1.00 P = 1.00Trembling H(ddl3) = 0.00; H(ddl3) = 0.00; P = 1.00 P = 1.00 DiarrhoeaH(ddl3) = 0.00; H(ddl3) = 0.00; P = 1.00 P = 1.00 Exophthalmos H(ddl3) =0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Piloerection H(ddl3) = 0.00;H(ddl3) = 0.00; P =1.00 P =1.00 Salivation H(ddl3) = 0.00; H(ddl3) =0.00; P =1.00 P =1.00 Lacrimation H(ddl3) = 0.00; H(ddl3) = 0.00; P=1.00 P =1.00 Palpebral reflex H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P=1.00 Strange behaviours H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00Bending the trunk H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Pelvicelevation H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Elevation ofthe tail H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00 Rectaltemperature H(ddl3) = 0.17; H(ddl3) = 3.67; P =0.98 P =0.30 Mortality at24 h H(ddl3) = 0.00; H(ddl3) = 0.00; P =1.00 P =1.00

TABLE 3 State of the variables before (T0) and after (T14)administration of AE for 14 days at the doses of 20, 40 and 80 mg/kg,p.o. FOB session T0 T14 Groups AE AE AE AE AE AE Items Carrier 20 40 80Carrier 20 40 80 Behaviour of the rat in H(ddl3) = 5.15; H(ddl3) =19.97; the housing cage P = 0.16 P = 0.0002 (D1** - D2** - D3**)Behaviour of the rat in H(ddl3) = 4.02; H(ddl3) = 7.19; the observationcage P = 0.26 P = 0.066 (D2# - D3*) Spontaneous H(ddl3) = 4.33; H(ddl3)= 15.20; locomotor activity in P = 0.23 P = 0.008 the housing cage(D1** - D2** - D3**) Interest for a shown H(ddl3) = 4.86; H(ddl3) =6.86; object P = 0.18 P = 0.08 (curiosity) (D1* - D2# - D3*) Flightreaction upon H(ddl3) = 2.39; H(ddl3) = 6.65; approaching a finger P =0.50 P = 0.08 (D1#- D2#- D3*) Reaction to pinching H(ddl3) = 5.50;H(ddl3) = 15.07; the tail P = 0.14 P = 0.002 (D1** - D2** - D3**)Reaction to pinching a H(ddl3) = 4.13; H(ddl3) = 9.63; hind paw P = 0.25P = 0.02 (D1** - D2# - D3#) Flight reaction to being H(ddl3) = 2.12;H(ddl3) = 8.01; touched P = 0.55 P = 0.045 (D1* - D2# - D3#) Cardiac andH(ddl3) = 4.60; H(ddl3) = 10.78; respiratory frequency P = 0.20 P = 0.01(D1# - D2** - D3**) Caused freezing H(ddl3) = 3.00; H(ddl3) = 6.27; P =0.39 P = 0.09 Muscular tonicity of the H(ddl3) = 3.83; H(ddl3) = 8.52;abdomen P = 0.28 P = 0.04 (D2# - D3*) Agitation-nervosity- H(ddl3) =1.04; H(ddl3) = 6.77; irritability P = 0.79 P = 0.08 (D2# - D3*) #P <0.10; *P < 0.05; **P < 0.01 (Mann-Whitney Test: vs. Carrier)

Comparisons of the Different Repeated Variables Between the FOBs at T0and T14

Daily administration of AE at certain doses for 14 days seems to have aneffect on certain studied main variables (Table 4):

-   -   The behaviour of the rats and their spontaneous locomotor        activity in the observation cage significantly increased under        AE at the 3 doses (stimulating effect or anti-stress effect),    -   The displacement latency of the control rats in the observation        arena increases and their locomotor activity (crossed spaces)        decreases, while in rats treated with AE, these variables remain        stable between the T0 and T14 FOBs (anti-stress and        antidepressant effects),    -   The interest for a shown object: at the dose of 20 mg/kg, p.o.        (per os), AE stimulates the interest of the rats for a stimulus        object which is presented to them (motivation for exploring        novelty, hedonism: anti-stress and antidepressant effects),    -   The number of crossed spaces on an open branch: comparatively        with the carrier, AE does not decrease the number of crossed        spaces on an open branch (anti-stress effect),    -   The flight reactions upon approaching the finger: AE at the 3        doses decreases the flight reaction upon approaching the finger        of the experimenter (anti-stress effect),    -   The reaction to pinching the tail increases in the control rat,        while under 20 mg/kg of AE it decreases (anti-stress effect        and/or antalgic effect),    -   The reaction to pinching a hind paw decreases under AE, at the 3        doses (anti-stress effect and/or antalgic effect),    -   The jump reaction to a sound stimulus significantly decreases at        the AE doses of 40 and 80 mg/kg (calming and anti-stress        effects),    -   The cardiac and respiratory frequencies decrease under AE at the        doses of 20 and 40 mg/kg (calming and anti-stress effects),    -   Agitation, nervosity and irritability: at the strong dose of 80        mg/kg, AE shows a positive effect on agitation, nervosity and        irritability of the rats (calming, anti-stress and/or        anti-aggressivity effects).

TABLE 4 Effects of AE on the time-dependent change of the variablesbetween the FOBs at T0 and T14 in each treatment group Groups CARR AE/20AE/40 AE/80 FOB session Items T0 T14 T0 T14 T0 T14 T10 T14 Behaviour ofthe rat N.S.

in the observation cage Spontaneous N.S.

locomotor activity in the observation cage Displacement latency

N.S. N.S. N.S. in the arena Locomotor activity

N.S. N.S. N.S. (spaces crossed) Interest for a shown N.S.

N.S. N.S. object Crossed spaces on an

N.S. N.S. N.S. open branch Flight reaction upon N.S.

approaching a finger Reaction to pinching

N.S. N.S. the tail Reaction to pinching a N.S.

hind paw Suspension test

N.S.

Jump reaction to a N.S. N.S.

sound stimulus Cardiac and N.S.

N.S. respiratory frequency Muscular tonicity of

N.S. N.S. N.S. the abdomen Agitation-nervosity- N.S. N.S. N.S.

irritability N.S.: Not significant between T0 and T14

 Decreases between T0 and T14

 Increases between T0 and T14 *P < 0.05; #: P < 0.10 (trend)

Elevated Plus Maze Test (LCS)

In the elevated-plus maze test, after 18 days of daily treatment, AE atthe doses of 20, 40 and 80 mg/kg, p.o., significantly increases thenumber of entries (Table 5, FIG. 4) as well as the spent time (Table 6,FIG. 5) of the rats treated in the open arms, demonstrating anti-stressactivity of AE.

TABLE 5 Effects of AE on the number of entries into the open arms in theelevated plus maze test AE 20 AE 40 AE 80 Carrier mg/kg mg/kg mg/kgKruskal- Products (n = 6) (n = 6) (n = 6) (n = 6) Wallis test Number ofentries 1.5 4.0 5.0 4.5 H(ddl3) = into the open (1.0- (4.0- (3.0- (2.0-6.08 branches Mean 4.0) 5.0) 5.0) 5.0) P = 0.108 (QI-QS)

TABLE 6 Effects of AE on the time spent in the open arms in theelevated-plus maze test AE 20 AE 40 AE 80 Carrier mg/kg mg/kg mg/kgKruskal- Products (n = 6) (n = 6) (n = 6) (n = 6) Wallis Test Time spentin the 10.0 37.5 50.5 37.0 H(ddl3) = open branches (7.0- (32.0- (38.0-(17.0- 7.76 Mean (QI-QS) 29.0) 50.0) 66.0) 45.0) P = 0.051

Aversive Light Stimulus Avoidance Response Test (ALSAR)

After 18 days of daily administration of AE, the Kruskal-Wallis testshowed that the number of cumulated pressing actions on both levers(active lever+inactive lever) of the rats of the different treatmentgroups are homogeneous at 5 minutes, 10 minutes, 15 minutes and at 20minutes (FIG. 6).

In the test for avoiding an aversive light stimulus, at the end of thefirst 5 minutes of the test, after 18 days of administration of AE, therats treated with the dose of 20 mg/kg, p.o., tend to discriminate theactive lever from the inactive lever (FIG. 7).

At the end of the period of 10 minutes of the test, the rats treatedwith AE at the dose of 40 mg/kg, p.o., significantly discriminate theactive lever from the inactive lever and those treated with doses of 20and 80 mg/kg, p.o., tend to operate a discrimination between bothlevers. This is not the case of the control rats which do not show anytrend towards discrimination (FIG. 8).

At the end of the period of 15 minutes of test, the rats treated with AEat the doses of 20 and 40 mg/kg, p.o., significantly discriminate theactive lever from the inactive lever. The rats treated at the dose of 80mg/kg, p.o., no longer discriminate both levers. The control rats stilldo not show any discrimination at this stage of the training test (FIG.9).

At the end of the period of 20 minutes of test, the rats treated with AEat the doses of 20 and 40 mg/kg, p.o., still discriminate significantlythe active lever from the inactive lever. The rats treated with the doseof 80 mg/kg, p.o., no longer show any discrimination and the controlrats begin to exhibit a beginning of discrimination (FIG. 10).

These results show the efficiency of AE on acquiring training, andnotably at the doses of 20 and 40 mg/kg, p.o.

Physiological and Innocuity Data

Administration of AE, regardless of the dose, did not show any influenceon the weight of the rats daily treated with the doses of 20, 40 or 80mg/kg, p.o., or on their food intake. No toxicity was detected inanimals treated with AE at the doses of 20, 40 and 80 mg/kg/d, p.o., for21 days.

Conclusion

The administration of an algal extract according to the presentinvention thus shows the neurological effects of the product, notablythe anti-stress/anxiolytic, anti-depressant, antalgic activities as wellas an early acquisition activity of training and of facilitatingmemorization.

Example 5: Evaluation of the Effects of the Algal Extract (AE) Throughthe Behavioural Despair Test

The anti-depressant effects of an algal extract according to theinvention (AE), administered orally as a preventive treatment with 3doses (10, 20 and 40 mg/kg/d) were evaluated in adult male Wistar ratsin the behavioural despair test (BDT).

The extract was daily administered as a semi-chronic treatment for 14days. The effects of the extract were evaluated in the BDT on groups of12 rats as compared with a control batch treated with the carrier(spring water) and a reference batch treated with Imipramine at the doseof 10 mg/kg/d.

The rats used were treated according to the ethical rules dictated byASAB and the Canadian Council for Animal Protection.

Equipment and Methods

Animals

Sixty male Wistar rats (Charles River Laboratories, France) weighingfrom 200-225 g were used. Upon receiving them, the rats were marked anddistributed in groups of 4 in polycarbonate cages of the F type(48×27×20 cm, U.A.R., 91-Epinay-Sur-Orge, France). The animals werehoused in an air-conditioned animal housing facility, at a temperatureof 22±2° C. and a relative humidity of 50±20%. The rats had standardfeed 2016 (Harlan, Gannat, France) and drink ad libitum. They weresubject to a reverse light-darkness cycle of 12 hours.

After getting used to the conditions of the laboratory for one week, therats were weighed and randomized according to their weight in 5treatment groups (n=12/group).

Treatment Groups:

Control group: treatment with spring water (Carrier),

Imipramine group: treatment with Imipramine at the dose of 10 mg/kg/d(Imi),

AE group 10: treatment with AE at the dose of 10 mg/kg/d (AE 10),

AE group 20: treatment with AE at the dose of 20 mg/kg/d (AE 20),

AE group 40: treatment with AE at the dose of 40 mg/kg/d (AE 40).

In order to avoid possible interferences between the differenttreatments, the rats of a same cage all receive the same dose of AE. Therats of the different groups are all handled in the same way and underthe same conditions.

Behavioural Despair Test (BDT)

The experimental devices consisted in Plexiglas cylinders with a heightof 50 cm and a diameter of 20 cm filled with water at 25° C. up to aheight of 30 cm.

After 13 days of daily treatment with the products to be tested, therats were placed at D14 in the devices for a period of 15 minutes inorder to evaluate the immobility time of each animal. Immediately afterthis first test, the rats received their 14^(th) treatment.

At D15, the rats received two treatments: the first, 5 hours before thetest and the second 1 hour before the test. This procedure is usuallyused in the literature for testing the efficiency of a product againstdepression. The test was conducted under the same conditions as the daybefore for a period of 5 minutes.

The recorded immobility duration during the first 5 minutes of bothtests (at D14 and D15) reflects the capacity of the animal of resigningitself when it is confronted with a situation from which it cannotescape. This resignation behaviour is assimilated with depression.

Product

The product is an AE (algal extract) as a powder representing afreeze-dried and crushed concentrated fraction of a water-soluble algalextract prepared according to Example no. 1. The extract to be testedwas dissolved in spring water and was administered to the rats orally atone of the 3 tested doses for 15 successive days with an administrationvolume of 5 ml/kg. The animals were weighed every two or three days inorder to adapt their specific treatments to their weight. Their food andwater intakes were noted every two or three days during the wholeduration of the study.

The animals of the Reference group receive daily 10 mg/kg/d ofImipramine hydrochloride (Sigma Aldrich) dissolved in spring water withan administration volume of 5 ml/kg.

The animals of the Carrier group daily received spring water with anadministration volume of 5 ml/kg.

Statistical Analysis

ANOVA in parametric mode was applied, followed, in the case ofheterogeneity, by comparisons by means of the non-paired t-test forcomparing the treated groups with the Carrier group and the Referencegroup.

Paired ANOVA was used, followed, in the case of heterogeneity, bycomparisons with the paired t-test for two by two comparisons ofrepeated measurements of BDT in each of the groups.

For the food and water intakes, non-parametric mode analysis wasapplied: a Kruskal-Wallis test if necessary followed by a Mann Whitney Utest for comparing the groups two by two.

The statistical and graphic processing operations were achieved by meansof the software package Statview5 (SAS Institute, Inc., USA).

Results

Behavioural Despair Test: Pre-Test at D14

At D14, in the pre-test of the behavioural despair, ANOVA did not showany heterogeneity for the immobility times of the animals of thedifferent treatment groups (Table 7; FIG. 11). This pre-test gives thepossibility of obtaining induction of resignation in the animals of thedifferent treatment groups.

TABLE 7 Effects of AE on the immobility time(s) in the behaviouraldespair pre-test at D 14 (s, Average ± SME) Imipramine AE AE AE Carrier10 mg/kg 10 mg/kg 20 mg/kg 40 mg/kg Products (n = 12) (n = 12) (n = 12)(n = 12) (n = 12) ANOVA_((ddl=4)) Immobility 18.7 ± 3.6 16.3 ± 4.6 12.5± 3.8 9.8 ± 3.2 9.3 ± 1.8 F = 1.33 duration (s) P = NS NS:non-significant.

Behavioural Despair Test at D15

At D15, in the behavioural despair test, ANOVA showed heterogeneity forthe immobility time of the animals of the different treatment groups(Table 8).

The non-paired t-test showed that the immobility times of the animalstreated with Imipramine, AE 20 and AE 40 were significantly shorter thanthat of the animals with the Carrier (t=3.69, P=0.0013; t=2.20, P=0.039and t=3.29, P=0.003, respectively).

The non-paired t-test showed that the immobility time of the animalstreated with AE 20 tended to be significantly longer than that of theanimals treated with Imipramine (t=1.72, P=0.099) (Table 8; FIG. 12).

TABLE 8 Effects of AE on the immobility duration(s) in the behaviouraldespair test at D 15 (s, Average ± ESM) Imipramine AE AE AE Carrier 10mg/kg 10 mg/kg 20 mg/kg 40 mg/kg Products (n = 12) (n = 12) (n = 12) (n= 12) (n = 12) ANOVA_((ddl=4)) Immobility 14.4 ± 2.7 3.8 ± 1.0 12.0 ±5.0 7.3 ± 1.8 4.9 ± 1.0 F = 2.76  duration (s) P = 0.036 Non-pairedt-test t = 3.69  t = 0.42 t = 2.20 t = 3.29 (vs. Carrier) P = 0.0013 NS P = 0.039  P = 0.003 Non-paired t-test t = 1.60 t = 1.72 t = 0.75 (vs.Imipramine) P = NS  T (P = 0.099)   NS Non-paired t-test t = 0.98 t =1.38 (vs. AE 10) NS NS Non-paired t-test t = 1.17 (vs. AE 20) NS NS: notsignificant.

Comparison of the Results of the Tests at D14 and D15

The paired t-test showed that the immobility times of the rats treatedwith Imipramine and AE 40 significantly decrease between D14 and D15(t=2.90, P=0.015 and t=2.42, P=0.034, respectively) (Table 9).

TABLE 9 Comparison of the results of the tests at D 14 and D 15 (s,Average ± SME) Imipramine AE AE AE Carrier 10 mg/kg 10 mg/kg 20 mg/kg 40mg/kg Products (n = 12) (n = 12) (n = 12) (n = 12) (n = 12) Immobilityduration (s) 18.7 ± 3.6 16.3 ± 4.6 12.5 ± 3.8 9.8 ± 3.2 9.3 ± 1.8 at D14 Immobility duration (s) 14.4 ± 2.7  3.8 ± 1.0 12.0 ± 5.0 7.3 ± 1.84.9 ± 1.0 at D 15 Paired t-test t= 1.41 2.90  0.11 1.10 2.42  P= NS0.015 NS NS 0.034 NS: not significant.

Weight Evolution, Food and Water Intakes

No effect of AE administered at the three doses was shown as regards theweight evolution and the food and water intakes of the animals.

Behavioural Despair Test

At D14: during the test for setting resignation in the animals, althoughthe immobility duration of the animals treated with AE at the doses of20 and 40 mg/kg is shorter than those of the animals of the othergroups, no significant difference was observed between the rats treatedwith the Carrier, Imipramine and AE administered at the three testeddoses.

At D15: the anti-depressant effect of AE administered at the doses of 20and 40 mg/kg was ascertained since the immobility durations of theanimals treated at these doses were significantly shorter than those ofthe animals treated with the Carrier. AE administered at the dose of 40mg/kg gave the possibility of obtaining an immobility duration similarto that of the animals treated with Imipramine at 10 mg/kg. Adose-dependent effect was observed in animals treated with AE at thedoses of 10, 20 and 40 mg/kg.

Comparison of the immobility durations at D14 and D15: only the animalstreated with Imipramine at the dose of 10 mg/kg and with AE at the doseof 40 mg/kg have significantly reduced their immobility duration betweenD14 and D15.

Behaviour of the Animals and Innocuity of the Algal Extract

No abnormal behaviour of the whole of the animals was observed duringthe experimentation for AE administered at the doses of 10, 20 and 40mg/kg. No toxicity was detected in animals treated with AE at the dosesof 10, 20 and 40 mg/kg/d for 14 days.

CONCLUSION

The administration of an algal extract according to the presentinvention thus demonstrates an anti-depressant effect of the product.

1. A method for treating and/or preventing neurological disorders,stress, anxiety, aging, Alzheimer's disease, neuropathic andinflammatory chronic pain, pain of the skin and/or of subcutaneoustissues and/or associated organs, or body pain, the method comprisingthe administration of an effective amount of an algal extract from theorder of Ulvales comprising sulphated and non-sulphated polyanionicpolysaccharides, the size of which is less than or equal to 50 kDa. 2.The method according to claim 1, wherein the extract is an extract ofgreen algae of the type Ulva.
 3. The method according to claim 1,wherein said polysaccharides comprise mannose and/or arabinose.
 4. Themethod according to claim 3, wherein said polysaccharides comprise atleast 0.005% of mannose and/or at least 0.005% of arabinose, by weightbased on the weight of the total dry material of the algal extract. 5.The method according to claim 4, wherein said polysaccharides comprisemannose at an amount in a range from 0.01 to 0.20% and arabinose at anamount in the range of 0.01 to 0.5%, by weight based on the weight ofthe total dry material of the algal extract.
 6. The method according toclaim 1, wherein said polysaccharides further comprise galactose,glucose, rhamnose, xylose, and glucuronic acid.
 7. The method accordingto claim 6, wherein: the galactose is at an amount in the range of 0.05to 0.5%; the glucose is at an amount in the range of 0.005 to 0.05%; therhamnose is at an amount in the range of 2 to 15%; the xylose is at anamount in the range of 0.1 to 1%; and the glucuronic acid is at anamount in the range of 1 to 7%; by weight based on the weight of thetotal dry material of the algal extract.
 8. The method according toclaim 1, wherein said polysaccharides have a size of less than or equalto 15 kDa.
 9. The method according to claim 1, wherein the algal extractcomprises: carbon at an amount in the range of 10 to 50%; hydrogen at anamount in the range of 1 to 10%; nitrogen at an amount in the range of 1to 5%; oxygen at an amount in the range of 20 to 50%; and sulphur at anamount in the range of 1 to 15%; in weight percentage of the total drymaterial of the algal extract.
 10. The method according to claim 1,wherein the algal extract comprises: carbon at an amount in the range of15 to 30%; hydrogen at an amount in the range of 3 to 6%; nitrogen at anamount in the range of 1 to 3%; oxygen at an amount in the range of 25to 40%; and sulphur at an amount in the range of 2.5 to 10%; in weightpercentage of the total dry material of the algal extract.
 11. Themethod according to claim 1, wherein the algal extract is characterizedby the ¹H NMR spectrum in FIG.
 1. 12. The method according to claim 1,wherein the algal extract is prepared by a process comprising thefollowing steps: a) washing the algae and freeing the algae of sand; b)crushing the washed and freed algae to create a crushed productcomprising a solid phase and a liquid phase; c) separating the solidphase of the crushed product from the liquid phase of the crushedproduct; d) clarifying the separated liquid phase to form a juice; e)filtering the juice with a membrane of less than 50 kDa; and f)concentrating the filtered juice; and g) drying the concentrated juice.13. The method according to claim 12, wherein membrane is a 15 kDamembrane.
 14. The method according to claim 12, wherein solid phase isseparate from the liquid phase by pressing the crushed product.
 15. Themethod according to claim 12, wherein: the algae is washed infreshwater; the algae are crushed with a refiner; the solid phase of thecrushed product is separated from the liquid phase of the crushedproduct by pressing the crushed product with a belt press; the separatedliquid phase is clarified with a clarifier with disc stacks; themembrane is a 15 kDa ceramic membrane; the filtered juice isconcentrated by evaporation; and/or the concentrated juice is dried byfreeze-drying or atomization.
 16. The method according to claim 12,wherein the composition further comprises for at least onepharmaceutically acceptable excipient.